Dataset Information

Characterization of the Tn916 conjugative transposon in a food-borne strain of Lactobacillus paracasei.

ABSTRACT: Food-borne antibiotic-resistant lactic acid bacteria have received growing attention in the past few years. We have recently identified tetracycline-resistant Lactobacillus paracasei in samples of milk and natural whey starter cultures employed in the manufacturing process of a typical Italian fermented dairy product, Mozzarella di Bufala Campana. In the present study, we have characterized at the molecular level the genetic context of tetracycline resistance determinants in these natural strains, which we have identified as tet(M). This gene was present in 21 independent isolates, whose fingerprinting profiles were distributed into eight different repetitive extragenic palindromic groups by cluster analysis. We provide evidence that the gene is associated with the broad-host, conjugative transposon Tn916, which had never before been described to occur in L. paracasei. PCR analysis of four independent isolates by use of specifically designed primer pairs detected the presence of a circular intermediate form of the transposon, carrying a coupling sequence (GGCAAA) located between the two termini of Tn916. This novel coupling sequence conferred low conjugation frequency in mating experiments with the recipient strain JH2-2 of Enterococcus faecalis.

SUBMITTER: Devirgiliis C

PROVIDER: S-EPMC2698359 | biostudies-literature | 2009 Jun

REPOSITORIES: biostudies-literature

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Characterization of the Tn916 conjugative transposon in a food-borne strain of Lactobacillus paracasei.

Devirgiliis Chiara C Coppola Doriana D Barile Simona S Colonna Bianca B Perozzi Giuditta G

Applied and environmental microbiology 20090424 12

Food-borne antibiotic-resistant lactic acid bacteria have received growing attention in the past few years. We have recently identified tetracycline-resistant Lactobacillus paracasei in samples of milk and natural whey starter cultures employed in the manufacturing process of a typical Italian fermented dairy product, Mozzarella di Bufala Campana. In the present study, we have characterized at the molecular level the genetic context of tetracycline resistance determinants in these natural strain ...[more]

PMID: 19395574

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Project description:Integrative and conjugative elements (ICEs), also known as conjugative transposons, are self-transferable elements that are widely distributed among bacterial phyla and are important drivers of horizontal gene transfer. Many ICEs carry genes that confer antibiotic resistances to their host cells and are involved in the dissemination of these resistance genes. ICEs reside in host chromosomes but under certain conditions can excise to form a plasmid that is typically the substrate for transfer. A few ICEs are known to undergo autonomous replication following activation. However, it is not clear if autonomous replication is a general property of many ICEs. We found that Tn916, the first conjugative transposon identified, replicates autonomously via a rolling-circle mechanism. Replication of Tn916 was dependent on the relaxase encoded by orf20 of Tn916 The origin of transfer of Tn916, oriT(916), also functioned as an origin of replication. Using immunoprecipitation and mass spectrometry, we found that the relaxase (Orf20) and the two putative helicase processivity factors (Orf22 and Orf23) encoded by Tn916 likely interact in a complex and that the Tn916 relaxase contains a previously unidentified conserved helix-turn-helix domain in its N-terminal region that is required for relaxase function and replication. Lastly, we identified a functional single-strand origin of replication (sso) in Tn916 that we predict primes second-strand synthesis during rolling-circle replication. Together these results add to the emerging data that show that several ICEs replicate via a conserved, rolling-circle mechanism.ImportanceIntegrative and conjugative elements (ICEs) drive horizontal gene transfer and the spread of antibiotic resistances in bacteria. ICEs reside integrated in a host genome but can excise to create a plasmid that is the substrate for transfer to other cells. Here we show that Tn916, an ICE with broad host range, undergoes autonomous rolling-circle replication when in the plasmid form. We found that the origin of transfer functions as a double-stranded origin of replication and identified a single-stranded origin of replication. It was long thought that ICEs do not undergo autonomous replication. Our work adds to the evidence that ICEs replicate autonomously as part of their normal life cycle and indicates that diverse ICEs use the same replicative mechanism.

Project description:BackgroundLactobacilli are often recognized as beneficial partners in human microbial environments. However, lactobacilli also cause diseases in human, e.g. infective endocarditis (IE), septicaemia, rheumatic vascular disease, and dental caries. Therefore, the identification of potential pathogenic traits associated with lactobacilli will facilitate the prevention and treatment of the diseases caused by lactobacilli. Herein, we investigated the genomic traits and pathogenic potential of a novel bacterial strain Lactobacillus paracasei LP10266 which has caused a case of IE. We isolated L. paracasei LP10266 from an IE patient's blood to perform high-throughput sequencing and compared the genome of strain LP10266 with those of closely related lactobacilli to determine genes associated with its infectivity. We performed the antimicrobial susceptibility testing on strain LP10266. We assessed its virulence by mouse lethality and serum bactericidal assays as well as its serum complement- and platelet-activating ability. The biofilm formation and adherence of strain LP10266 were also studied.ResultsPhylogenetic analysis revealed that strain LP10266 was allied with L. casei and L. paracasei. Genomic studies revealed two spaCBA pilus clusters and one novel exopolysaccharides (EPS) cluster in strain LP10266, which was sensitive to ampicillin, penicillin, levofloxacin, and imipenem, but resistant to cefuroxime, cefazolin, cefotaxime, meropenem, and vancomycin. Strain LP10266 was nonfatal and sensitive to serum, capable of activating complement 3a and terminal complement complex C5b-9 (TCC). Strain LP10266 could not induce platelet aggregation but displayed a stronger biofilm formation ability and adherence to human vascular endothelial cells (HUVECs) compared to the standard control strain L. paracasei ATCC25302.ConclusionThe genome of a novel bacterial strain L. paracasei LP10266 was sequenced. Our results based on various types of assays consistently revealed that L. paracasei LP10266 was a potential pathogen to patients with a history of cardiac disease and inguinal hernia repair. Strain LP10266 showed strong biofilm formation ability and adherence, enhancing the awareness of L. paracasei infections.

Project description:Lactic acid bacteria (LAB) are associated with various plant, animal, and human niches and are also present in many fermented foods and beverages. Thus, they are subjected to several stress conditions and have developed advanced response mechanisms to resist, adapt, and grow. This work aimed to identify the genes involved in some stress adaptation mechanisms in LAB. For this purpose, global reverse genetics was applied by screening a library of 1287 Lactobacillus paracasei transposon mutants for mild monofactorial stresses. This library was submitted independently to heat (52°C, 30 min), ethanol (170 g.L-1, 30 min), salt (NaCl 0.8 M, 24 h), acid (pH 4.5, 24 h), and oxidative (2 mM H2O2, 24 h) perturbations which trigger mild monofactorial stresses compatible with bacterial adaptation. Stress sensitivity of mutants was determined either by evaluating viability using propidium iodide (PI) staining, or by following growth inhibition through turbidity measurement. The screening for heat and ethanol stresses lead respectively to the identification of 63 and 27 genes/putative promoters whose disruption lead to an increased sensitivity. Among them, 14 genes or putative promoters were common for both stresses. For salt, acid and oxidative stresses, respectively 8, 6, and 9 genes or putative promoters were identified as essential for adaptation to these unfavorable conditions, with only three genes common to at least two stresses. Then, RT-qPCR was performed on selected stress response genes identified by mutant screenings in order to evaluate if their expression was modified in response to stresses in the parental strain. Eleven genes (membrane, transposase, chaperone, nucleotide and carbohydrate metabolism, and hypothetical protein genes) were upregulated during stress adaptation for at least two stresses. Seven genes, encoding membrane functions, were upregulated in response to a specific stress and thus could represent potential transcriptomic biomarkers. The results highlights that most of the genes identified by global reverse genetics are specifically required in response to one stress and that they are not differentially transcribed during stress in the parental strain. Most of these genes have not been characterized as stress response genes and provide new insights into the adaptation of lactic acid bacteria to their environment.

Dataset Information

Characterization of the Tn916 conjugative transposon in a food-borne strain of Lactobacillus paracasei.

Publications

Characterization of the Tn916 conjugative transposon in a food-borne strain of Lactobacillus paracasei.

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